Long term changes in gene expression are believed to contribute importantly to the mechanisms underlying drug addiction. Considerable attention has been devoted to the cAMP second messenger pathway because it is upregulated by opiates and other drugs of abuse. Both tolerance and dependence have been attributed to changes in function of the transcription factor CREB, which mediates cAMP-dependent gene expression. It is currently believed that CREB binds constitutively to a promoter element termed the CRE. This proposal challenges this model and tests a new hypothesis-that chronic exposure to morphine induces CREB binding to some genes but not others. To address this hypothesis the lab has developed a novel approach termed SACO for examining CREB binding to target genes in vivo. This method combines chromatin immunoprecipitation with a modification of Long SAGE (an approach designed for analysis of mixtures of RNA). SACO will be used to identify the entire complement of CREB targets and measure how the selection of these targets is affected by agents, such as morphine, that upregulate the cAMP pathway. Additional studies will address the mechanisms underlying the morphological changes in dendritic processes induced by CREB that occur after treatment with other drugs of abuse, namely cocaine and amphetamine. Specific goals of this project are to identify the entire set of CREB targets in human neuroblastoma cells and determine whether this set is altered by acute exposure to cAMP or chronic exposure to morphine. Previous studies have indicated that CREB regulates the expression of both protein-coding and noncoding transcripts. Many protein-coding transcripts and most noncoding transcripts are missing from conventional microarrays, however. Studies in this proposal will examine both classes of RNAs by developing a custom microarray representing the CREB transcriptome. One specific microRNA, designated miR132, was found to be induced by CREB in PC12 cells and to stimulate changes in dendritic morphology in neurons that are highly reminiscent of those caused by CREB activators. A candidate target for this miRNA has been identified and experiments are designed to elucidate the mechanism of miR132 action. The concept that CREB signaling induces mediators of translational arrest has profound implications for the understanding of drug action.